| Establishment of popla（rPopulus tomentosa）and ectomycorrhizal fung（iBoletus edulis）hydroponic interaction experiment system and collection the signal liquid containingwater-soluble signal factors to detect the innate immunity response characteristics and secondmessenger transmembrane transport of host plant roots during the presymbiotic stage. Focusingon analysis of cascade relationship between Ca2+and hydrogen peroxide(H2O2) transmembranetransport and symbiosis related genes, discussion the startup mode about host plants earlyimmune mechanism during the presymbiotic stage of ECM. The main results and conclusionsare as follow:1. Design two kinds of hydroponic interaction experiment system to simulate signalsconduction events between fungi and plant in the early stage of ECM symbiosis, analysis theeffect of symbiotic signals on the candidate symbiosis-related genes expression. In thesimulation system of ECM fungi infection in host plant roots, the expression profiles of SOD,POD, PAL, APX and CIPK2showed significantly rising trend in5days, indicating that ECMfungi direct contact on host roots, will be quickly induced the transcriptional expression ofthese genes. While in the simulation system of signals transduction, genes expression patternsof SOD, POD, PAL and APX showed slowly rising state in plant roots, but these trends wereconsistent with the fungi infection system, in addition to APX, the transcription levels of otherthree genes all showed significantly increased during the interaction process, and the mRNAcontent of SOD, POD and APX reached the highest value in the interaction of the third day.The expression profile of CIPK2kept a significantly up-regulated state in the interactionprocess, which reached the peak after the first day and significantly higher than the control andthe simulation system of fungi infection, proved that ECM fungi effectively induced theexpression of symbiotic related genes in plant roots through secreted water-soluble signalfactors, and preliminary verified the effectiveness of signaling simulation system to studysymbiosis signaling events during the ECM prosymbiotic stage. In addition, through a comprehensive comparative analysis, on the third day after interaction, considered that thesymbiotic signals secreted by ECM fungi can effectively induce symbiosis-related genesexpression for plant roots in this system.2. Preparated and collected symbiosis signal liquid based on signal transductionsimulation system, as well as collected aqueous solutions of roots exudates and fungi exudates,contrasted analysis the impact on symbiosis-related genes expression in these treatment. Rootexudates have no significant effect on the expression of SOD、POD and CIPK2, but theexpression of these genes induced by fungi exudates showed significantly increased andreached the highest value in the interaction of12h. Symbiotic signals have the mostsignificantly induction for root genes expression, and which induced the most rapidly changesin transcription level of CIPK2, respectively significantly induced up-regulated expression in3h and9h to form a “double peak” showed continued upregulated. SOD reached two peaks inthe6h and12h, respectively, and its transcription level in12h achieved the highest value, PODup-regulated expression of the whole process showed significantly and rapidly trend. Throughthe contrast analysis that up-regulated expression of the roots immune genes can be induced byECM fungi secretion, but in the recognition process, fungi or roots through communicationsignal factors to sense the presence of each other, and further secret of soluble signalingmolecules to expand pre-symbiotic interaction phase of signal transduction, at this time, hostplant roots will improve the expression of immune response genes and initiative their earlyimmune mechanisms after perceived fungi source signaling molecules.3. By means of scanning ion-selective electrode technique to research dynamic changes ofCa2+transmembrane transport in root organizations induced by symbiotic signal factors. TheCa2+flow on root elongation zone showed a small inflow state in a stable environment, but thehuge outflow occured when was instantaneous treated with signals solution, and graduallystabilized at a small outflow of state. With roots were induced by signal factors, Ca2+transmembrane transport flows gradually transformed into a stable inflow state, and the flowrate increased with time, while the control is gradually returned to a steady small inflow state.In addition, under the instantaneous treated of fungi secretions and root secretions, the formation of Ca2+transmembrane transport was consistent with signals solution, while thelatter was gradually restored to the small inflow state like control, suggested that the symbioticsignals solution contained some kinds of signal molecules which induced Ca2+internal flowacross the membrane in roots epidermal cells, or taked the Ca2+as second messenger toregulate Ca2+influx after identified fungi signals, finally transducted the symbiotic relatedsignals in the plant body.4. Researched the dynamic changes of H2O2production mode in root organizationsinduced by symbiotic signal factors through scanning ion-selective electrode technique. H2O2showed a stable state in equilibrium conditions, while the root released large amounts of H2O2under different transient treatments, and gradually moving to small absorbed state at last. Withthe signal factors inducted, the level of it released H2O2were also rising and reached themaximum when interacted at9hours. Fungi secretions induced root produced H2O2within24hours, but root secretions inhibited the release on the whole which showed the balance orabsorbed state except released at a very lowly level in3hours. This experiment firstlydemonstrated for the dynamic mode of H2O2generated induced by signal factors in plant roots.5. By means of drug pharmacology experiments studied the action of H2O2and Ca2+signals in the early immune response, and explored the initiation mode of immune in ECMpresymbiotic stage. Pharmacological experiments demonstrated that the Ca2+outflow and H2O2production were effectively inhibited by LaCl3(plasma membrane calcium channel inhibitor),effectively inhibited Ca2+efflux host roots and H2O2generation, so that the root tissues havebeen in a absorption state for Ca2+and H2O2. While the DPI (plasma membrane NADPHoxidase inhibitors) could only effectively inhibited the production of H2O2in host roots, didnot affect the dynamic mode for Ca2+transmembrane transport, only reduced the speed of Ca2+outflows across membrane in3hours. In terms of gene expression profiles, the transcriptionlevel of CIPK2, SOD and POD genes were significantly inhibited when host plant roots betreatmented by LaCl3. But by means of DPI only inhibited the transcription levels of SOD andPOD genes, did not affect the normal expression of CIPK2genes. Thus speculated that signalfactors induced the Ca2+inflowed host roots across membrane during the prosymbiotic stage, and combined with CIPK2gene to regulate roots produced a large number of H2O2, in order toinduce transcriptional expression of immune related genes and initiative the host plants earlyimmune mechanisms. |
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